A modified “wound healing” model – human dental pulp stem cells cultured in a hypoxic environment

Authors

Schuyler Tang, University of the Pacific
Isabel Kwon, University of the Pacific
Marie Tolarova, University of the Pacific
Miroslav Tolar, University of the Pacific

Lead Author Affiliation

Dental Surgery Program

Introduction/Context/Diagnosis

Tissue regeneration during wound healing resembles, in some aspects, tissue formation during prenatal development. In particular, tissue regeneration and tissue prenatal development are sensitive to oxygen supply. It is intriguing that developmental anomalies such as cleft lip and palate and congenital heart defects may result from a local prenatal transient lack of oxygen supply. Thus, an in vitro study of “wound healing” in hypoxic environment may have implications not only for tissue recovery after traumatic injury, but also for pathogenesis of developmental abnormalities.

We are interested in using dental pulp stem cells (DPSCs) exposed to severe hypoxia as a model for development of craniofacial anomalies. DPSCs are appropriate for this task, because they share the common neural crest cell origin with tissues forming the maxilla.

Methods/Treatment Plan

Using a disposable scalpel blade, a sterile silicone foil (Grace Biolabs) was hand-cut into strips 2 mm, 3 mm, and 4 mm wide and 5-6 mm long. The strips were transferred into petri dishes (Falcon, 35 mm diameter): one pair of plates had only 2 mm strips, one pair had only 3 mm strips, and one pair had only 4 mm strips. One plate from each pair was used as a control, another plate was experimental. All the plates were seeded with DPSCs (1 million cells per dish, Celprogen) in alpha MEM supplemented with 10% human serum, L-glutamine and penicillin/streptomycin mixture and incubated at 37.0ºC, 5.0% CO₂, 99% humidity, normoxia (21% O₂) for 2 days. After 2 days, growth medium was changed, silicone strips were removed, and all plates were incubated for another 12 hours. Then, the three experimental plates were placed in a hypoxic chamber (Biospherix) with 0.5% O₂ for 6 hours before being returned to normoxia (21% O₂). Three control dishes were incubated only in normoxia (21% O₂). Pictures of selected fields of control and experimental cultures were taken every 24 hours for another three days (40x magnification). Image J (NIH/NIDCR) will be used for a quantitative evaluation of gaps’ areas covered by cells.

Results/Outcome

Through culturing of DPSCs, two cell phenotypes became apparent: round-shaped and spindleshaped. Under normal conditions, darker, spindle-shaped cells dominated cultures. During 12 hours after removal of silicone strips, the cells slightly advanced into the gap space from the margin. After three days of cultivation of the control plates, spindle-shaped cells predominated in culture and started filling the gap, while round-shaped cells spread throughout the dish. In the plates exposed to hypoxia for 6 hours and then cultivated for three days, cells in the monolayer outside of the gap margin were mostly round-shaped and a mass detachment was seen after 48 hours, while cells immediately inside of the gap margin were spindleshaped and exhibited apparent migration and proliferation. A degree, to which normoxia and hypoxia affected closure of the gaps has not yet been quantified.

Significance/Conclusions

We have established the basic conditions under which a “wound healing” model can be assembled, followed and evaluated. Further study is warranted to quantitatively determine the effects of hypoxia on DPSCs and to characterize the round-shaped and spindle-shaped cell phenotypes in more detail.

Comments/Acknowledgements

This pilot study was supported by DRES03

Location

University of the Pacific, Dugoni Dental School, San Francisco, CA

Format

Poster

Poster Session

1st and 2nd Year Student Research Presentations